Our overall Goal is to develop our bactericidal R-type pyocin targeting E.coli O157:H7, as a specific therapeutic against infection and contamination. Enterohemorrhagic E. coli (EHEC) are a group of food- and waterborne pathogens that cause illnesses ranging from non-bloody diarrhea to hemolytic uremic syndrome (HUS). E. coli O157:H7 causes both large outbreaks as well as sporadic infections, and thus poses a threat to public health. The work proposed here is based on our findings that R-type pyocins are highly specific and highly potent bactericidal protein complexes that have been shown to be efficacious for systemic bacterial infections in animals. While highly specific to their target organism, these bactericidal agents can be retargeted to other bacteria by modifying their tail fibers with the related tail proteins of bacteriophages. Our leading product candidate, AvR2-V10, consists of the tail fiber of the bacteriophage ?V10 fused to an R2-type pyocin, such that the chimeric particle has the bactericidal specificity against all tested O157 E. coli strains - i.e. the specificity of the bacteriophage. In the work proposed here, we will advance the development of AvR2-V10 as an anti E. coli O157:H7 agent. We hypothesize that R-type pyocins can be synthesized and produced in an E. coli BL21 system, yielding an effective therapeutic against E. coli O157:H7 infection. In order to achieve our goals, we will conduct preclinical testing of AvR2-V10 by producing it in a regulatable manner in E. coli BL21, such that it abides to GLP standards (Specific Aim #1). We will evaluate its efficacy in an infant rabbit model which mimics the human pathology associated with O157:H7 infection (Specific Aim #2). The product candidate is intended to be used as a therapeutic or prophylactic that specifically targets E.coli O157 and can offer a potent response in cases of epidemic, either accidental or intentional mass infection. PUBLIC HEALTH RELEVANCE: E. coli O157 can cause severe intestinal infections and potentially death, and poses a threat to public health in the form of a bioterrorist attack or accidental food or waterborne outbreak. We have used our platform technology to engineer a potent bactericidal agent, AvR2-V10, to specifically target E.coli O157. This agent is now being developed as a therapeutic or prophylactic against E.coli O157 infection or contamination, offering a potent response in cases of epidemic. Herein, we propose to develop a simple, high yield production method for this agent, and test it's efficacy in a rabbit model.